Recombinant adeno-associated virus has been shown to efficiently transduce a variety of tissues in vivo, which results in persistent expression of transgene products. Its general application in the treatment of genetic disorders, however, is limited by difficulties in achieving sufficiently high titers of the vectors. In this application we will address this limitation twofold: We have collected evidence that packaging may require additional cis-elements within the viral genome. We propose to map and characterize these potential signals and to incorporate them into rAAV genomes. Using standard methods for rAAV production we will evaluate the effect of these potential cis- acting signals on viral titers. To test our hypothesis we will generate a number of deletion mutants of pAV2 (wtAAV). These mutants will be tested for genome replication in cell-free systems as well as for packaging in tissue culture in the presence of helper plasmids. A well-accepted hypothesis is that a rate-limiting step in recombinant AAV production is at the level of capsid (cap) expression in the producer cell. This limitation can be resolved by providing cap-genes in a construct which is capable of replicating in the producer cells but which is devoid of packaging signals. We propose to test the hypothesis that the ITRs of a different parvovirus can be used to achieve this goal. The goal of this aim is to provide rep and cap-genes in a construct, which is capable of replicating in the producer cells but which is devoid of packaging signals. We propose to test the hypothesis that the ITRS of a GPV can be used to achieve this goal. We have generated a chimeric Rep protein consisting of the GPV Rep1 origin interaction domain and the rest of AAV Rep78. Using this approach we were able to redirect the origin specificity of Rep78 from the AAV origin to the GPV origin. We propose to test if this protein is capable of mediating replication in tissue culture of a cap-containing construct, which is flanked by the GPV ITRs. Furthermore, we will test if genomes flanked by GPV ITRs lack the packaging signals for AAV capsids.